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-   -   Pre-Hybridization PCR not running efficiently (http://seqanswers.com/forums/showthread.php?t=90523)

MollySchools 08-15-2019 01:49 PM

Pre-Hybridization PCR not running efficiently
 
Hello all,
I am working on building my libraries for an NGS project. I am running into a problem in which my pre-amplified libraries have concentrations ranging from 8 ng/uL up to 15 ng/uL. However, when I attempt to amplify these libraries using a Phusion master mix and the primers that are compatible with our adapters for a 12 cycle PCR, the product qubits much lower (.5 ng/uL - 1 ng/uL) and appears to have not amplified. Running a normal QC PCR however I see product on my PCR gel indicating that the adapters ligated properly and are working with the primers. If anyone has any idea what my be causing this or possible troubleshooting steps that would be much appreciated!
Molly

luc 08-15-2019 04:53 PM

What type of libraries are you generating? How are you measuring concentrations?

I would suggest to avoid Phusion for complex libraries. It is fine for amplicon sequencing. Phusion has been shown to introduce comparatively strong biases according to GC content and fragment lengths. Usually, NEB Q5 or Kapa Hifi are used instead.

12 PCR cycles seems way too much, when starting out with 15 ng/ul. It could be possible that the libraries are overamplified, and thus are mostly single-stranded leading to low measurements.

MollySchools 08-19-2019 06:21 AM

Hi Luc,
Thank you for your reply. I have also tried to use Kapa Hifi and have seen the same results. I am using a qubit to measure concentrations. While 12 cycles may be to much for the samples that are 15 ng/uL I am observing the same results in libraries that are closer to 1 ng/uL as well. I will try to run my samples for less cycles and see if there is a noticeable difference. Thank you,
Molly


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